VaNAC26通过茉莉酸信号通路调控山葡萄耐旱性的机理研究

Improvement of cold and drought tolerance is one of the important goals in our grape industry. Vitis amurensis was widely used as one of the important resources during grape breeding to improve the cold and drought tolerance. But its drought tolerance related mechanisn is still far from understanding. In our previous work, the expression level of NAC26 gene was found accumulated quickly and largely during drought treatment in V. amurensis (VaNAC26) than in ‘Muscat of Hamburg’. VaNAC26- over-expressed Arabidopsis show increased drought tolerance than wildtype Arabidopsis. Transcriptome analysis show that Jasmonic acid (JA) synthesis and its downstream signaling related genes were up-regulatd in transgenic lines when compare with wildtype plants. The content of JA were found increased in VaNAC26- over-expressed line than wildtype Arabidopsis.While in V. amurensis we also found that JA accumulated during drought treatment. Based on these results, a series of experiments including suspension cell transformation, yeast one-hybrid/two hybrid, ChIP-Seq and transcriptome analysis were designed. The up-stream genes VaNAC26 will be identified to elucidate the possible reasons for its especial expression pattern under drought stress in V. amurensis. The proteins that may interact with VaNAC26 will be identified and the way of VaNAC26 regulated the expression of JA synthesis related gene will be analyzed. The aim of this study is to explain the mechanism that VaNAC26 participate in the drought stress response in V. amurensis by regulating the JA related signaling pathway. This study can increase our understanding of drought stress related signaling pathway in V. amurensis. The findings in this study will provide clue for better utilize V. amurensis resource and provide the drought tolerance related molecular makers for marker-assisted breeding.

提高葡萄的耐寒旱性是我国葡萄产业亟待解决的重要问题。山葡萄是我国葡萄耐寒旱育种的重要资源，但其耐旱机理仍不清楚。申请人在前期工作中发现NAC26基因在山葡萄中（VaNAC26）对干旱处理的响应要显著高于‘玫瑰香’葡萄；拟南芥中过表达VaNAC26可增加植株的耐旱性；转录组分析表明过表达植株中JA合成及下游信号通路相关基因的表达量增加；过表达植株中JA含量显著高于野生型；而山葡萄JA含量在干旱处理下亦明显增加。在此基础上，拟利用悬浮细胞转化、酵母单/双杂交、ChIP-Seq、转录组测序等技术，挖掘其特异表达模式的成因，鉴定其互作蛋白，并明确其对JA合成通路的调控方式，最终解释VaNAC26在山葡萄中如何通过调控JA信号通路来参与干旱应答。本研究对揭示山葡萄耐旱应答网络特点和调控机制具有重要理论意义，研究结果也将为充分利用山葡萄资源、开发新的耐寒旱分子标记用于辅助育种等工作提供分子元件。

提高葡萄的抗寒旱性是我国葡萄产业的迫切需求。山葡萄具有极强的抗寒旱能力，但是其抗性机理仍不清楚。前期研究结果表明VaNAC26能够在山葡萄中快速应答干旱胁迫并具有增强拟南芥抗旱性的功能。在此基础上，本项目以山葡萄和抗旱性弱的欧亚种‘玫瑰香’葡萄为研究材料，挖掘VaNAC26上下游调控因子，鉴定VaNAC26的互作蛋白并验证其功能，解释VaNAC26快速应答干旱的原因并初步揭示VaNAC26参与干旱应答的调控机理。主要研究结果如下：发现NAC26在山葡萄和‘玫瑰香’休眠芽中的表达模式相近；明确了VaNAC26启动子上游80bp的插入片段参与此基因的表达调控；以插入片段为诱饵，得到VaZHD7 等 5 个候选调控基因，并对VaZHD7可能结合的基序进行了突变验证；从3个候选基因中发现VaNAC17可以和VaNAC26在酵母和烟草中互作，并在拟南芥中证实VaNAC17参与植物抗旱调控；利用酵母系统鉴定了VaNAC17的转录激活区域，发现其表达受低温、干旱和高盐多种胁迫诱导；DAB和NBT染色证实过表达VaNAC17增加了植物的氧自由基清除能力并提高了植株的叶绿素含量；通过对过表达VaNAC17的拟南芥株系的转录组分析发现，正常条件下转录组变化不显著，干旱后胁迫相关基因如LEA等显著上调，茉莉酸合成基因表达增加，茉莉酸含量上调，表明VaNAC17可能通过与VaNAC26相似的信号通路来参与山葡萄干旱应答，并推测正常条件下，VaNAC17独自无法激活茉莉酸合成通路，需要VaNAC26同源基因相互作用共同促进茉莉酸合成基因的表达；通过共表达分析以及过表达VaNAC26葡萄愈伤的转录组分析，发现了5个参与茉莉酸合成的基因（AOC、AOS、LOX、MYC和OPR）在过表达愈伤中显著上调表达，并发现其启动子区含有NAC基因可以结合的NACRS元件，可能作为VaNAC26下游潜在靶基因来调节茉莉酸的含量；正在继续利用葡萄发根转化系统，对NAC26和NAC17的下游基因继续开展挖掘和验证。项目执行期间共发表SCI论文4篇，培养博士1名，硕士2名。本研究初步鉴定了VaNAC26在山葡萄抗旱应答中的上下游信号及互作蛋白的功能，为揭示山葡萄的抗旱性提供了理论依据。